8 - testing technology pt2 Flashcards
What are the different testing methods of pgx testing?
Testing the known variants:
- Genotyping (DNA chip)
Testing both known and unknown alleles:
- Sequencing (Sanger sequencing; high throughput nextgen sequencing)
What is the most useful technique for DNA amplification of 50-1000 bp?
Polymerase chain reaction (PCR)
What does PCR do?
Amplifies a specific region from genome for making billions of copies (detectable) (about 2^35)
What type of reaction is PCR?
Enzymatic rxn
What are the substrates of PCR?
- DNA template
- dNTPs (dATP, dGTP, dCTP, dTTP)
- Primers: 2 short sequences specific to region of interest
- Buffer: pH, Mg2+
- Enzyme: Taq DNA polymerase
What is the product of PCR?
DNA molecules (fragments start and end w primers)
How many copies of DNA does PCR make?
From 2 copies to 2^(n+1) copies [n = number of thermal cycles]
What amount of template DNA does PCR start w?
Starts from very small amount of template DNA (5-20 ng)
What is Taq polymerase enzyme?
- Key to PCR
- Thermal stable
- Isolated from Thermus aquaticus by Thomas D. Brock in 1965
Where does PCR amplify DNA from?
Both DNA molecules of homologous chromosomes
What are PCR reaction products?
Amplicon:
- Mixture of double strand DNA products generated from both homologous chromosomes (the primers equally bind to each chromosome)
What is DNA chip used for?
To detect known SNPs or targeted SNPs
What is high in DNA chip?
High throughput:
- Up to 5m SNPs can be genotyped simultaneously
What are elements of conventional sequencing?
- aka Sanger sequencing
- Low throughput
- Targeted sequencing: sequencing one specific DNA fragment
What are elements of next generation sequencing?
- High throughput sequencing
- Parallel sequencing
- Massive sequencing: sequencing multiple DNA fragment simultaneously
